Nucleic acid ligand diagnostic biochip

ABSTRACT

A diagnostic biochip comprising a solid support to which one or more specific nucleic acid ligands is attached in a spatially defined manner is provided. Each nucleic acid ligand binds specifically and avidly to a particular target molecule contained within a test mixture, such as a bodily fluid. Also provided are methods for the preparation of nucleic acid ligand biochips. Further, methods for the use of the nucleic acid ligand biochip in diagnosis of a medical condition and quantitative detection of a target molecule are provided.

This application is a 35 U.S.C. § 371 national phase application ofInternational Application No. PCT/US98/26515, published as InternationalPublication No. WO99/31275. PCT/US98/26515 is a continuation and claimspriority to U.S. patent application Ser. No. 08/990,436, filed Dec. 15,1997, now U.S. Pat. No. 6,242,246 issued Jun. 5, 2001.

FIELD OF THE INVENTION

The invention is directed to methods for the detection of targetmolecules in test solutions, particularly medically relevant moleculescontained in bodily fluids. The methods described herein use specificnucleic acid ligands attached to solid supports at spatially discretelocations. The invention provides methods for detecting the binding oftarget molecules to nucleic acid ligands, and methods for using arraysof nucleic acid ligands in diagnostic medical applications.

BACKGROUND OF THE INVENTION

A method for the in vitro evolution of nucleic acid molecules withhighly specific binding to target molecules has been developed. Thismethod, Systematic Evolution of Ligands by Exponential Enrichment,termed the SELEX process, is described in U.S. patent application Ser.No. 07/536,428, entitled “Systematic Evolution of Ligands by ExponentialEnrichment,” now abandoned, U.S. patent application Ser. No. 07/714,131,filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No.5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17,1992, entitled “Methods for Identifying Nucleic Acid Ligands,” now U.S.Pat. No. 5,270,163 (see also WO 91/19813), each of which is hereinspecifically incorporated by reference. Each of these applications,collectively referred to herein as the SELEX patent applications,describes a fundamentally novel method for making a nucleic acid ligandto any desired target molecule.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of nucleic acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound nucleic acids from those nucleic acidswhich have bound specifically to target molecules, dissociating thenucleic acid-target complexes, amplifying the nucleic acids dissociatedfrom the nucleic acid-target complexes to yield a ligand-enrichedmixture of nucleic acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific, high affinity nucleic acid ligands tothe target molecule.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, abandoned in favor of U.S. patentapplication Ser. No. 08/430,709, filed Apr. 27, 1995, now U.S. Pat. No.5,660,985, entitled “High Affinity Nucleic Acid Ligands ContainingModified Nucleotides,” that describes oligonucleotide containingnucleotide derivatives chemically modified at the 5- and 2′-positions ofpyrimidines. U.S. patent application Ser. No. 08/134,028, filed Oct. 7,1993, abandoned in favor of U.S. patent application Ser. No. 08/443,957,filed May 18, 1995, now U.S. Pat. No. 5,580,737, entitled “High-AffinityNucleic Acid Ligands That Discriminate Between Theophylline andCaffeine,” describes highly specific nucleic acid ligands containing oneor more nucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F),and/or 2′-O-methyl (2′-OMe). U.S. patent application Ser. No.08/264,029, filed Jun. 22, 1994, now abandoned, entitled “Novel Methodof Preparation of Known and Novel 2′ Modified Nucleosides byIntramolecular Nucleophilic Displacement,” describes oligonucleotidecontaining various 2′-modified pyrimidines.

Given the remarkable ability of nucleic acid ligands to be generatedagainst many different target molecules, it would be desirable to havemethods for using said ligands as a diagnostic tool. In particular, itwould be desirable to attach a plurality of different nucleic acidligands to a microfabricated solid support (a “biochip”), and then assaythe binding to said ligands of target molecules in a bodily fluid. Thesubject application provides such methods.

SUMMARY OF THE INVENTION

Methods are provided in the instant invention for obtaining diagnosticand prognostic nucleic acid ligands, attaching said ligands to abiochip, and detecting binding of target molecules in a bodily fluid tosaid biochip-bound nucleic acid ligands. In one embodiment of theinstant invention, one or more nucleic acid ligands are chosen that bindto molecules known to be diagnostic or prognostic of a disease; theseligands are then attached to the biochip. Particular methods forattaching the nucleic acid ligands to the biochip are described below inthe section entitled “Fabrication of the Nucleic Acid Biochip.” Thebiochip may comprise either (i) nucleic acid ligands selected against asingle target molecule; or more preferably, (ii) nucleic acid ligandsselected against multiple target molecules. In the subject invention,the level of target molecule binding to nucleic acid ligands at definedspatial locations will be determined using bodily fluid from individualsknown to have the disease for which that target molecule is known to beprognostic or diagnostic, and also using bodily fluid from healthyindividuals. Bodily fluid from an individual seeking a prognostic reportcan then be assayed using the biochip, and comparison of the three setsof data will yield prognostic or diagnostic information for thatindividual.

In another embodiment, the specific nucleic acid ligands attached to thebiochip bind specifically to all or a large number of components ofblood plasma, or other bodily fluids, of a healthy individual. Thepattern and level of binding of these ligands to their targets will thenbe determined by the methods disclosed below for healthy individuals,and also for individuals diagnosed with various medical conditions. Acomputer database of biochip binding data will then be established, witheach disease giving rise to a unique “signature” binding pattern. Bodilyfluids from individuals desiring a prognostic or diagnostic report willthen be contacted with the biochip, and the binding pattern obtainedcompared with the reference database.

In a related embodiment, the attached nucleic acid ligands will bindspecifically to all or a large number of components of the blood plasma,or other bodily fluid, of an individual known to be suffering from aparticular disease. Once the pattern and level of target moleculebinding to this biochip has been determined for the individual sufferingfrom the disease, this biochip can be used to screen individuals knownto be at risk of developing this disease. This embodiment will be usefulfor diseases in which the target molecules are not found in the bodilyfluid of healthy individuals, and the target molecules themselves arenot yet identified (e.g., for viral, bacterial or parasitic infectionswhere the causative agent has not yet been characterized at themolecular level).

In all the methods described in this invention, it is not necessary toknow what each nucleic acid ligand is binding. The preceding twoembodiments are particularly preferred, as they will allow for the earlydiagnosis of diseases for which there are no currently known assays, andfor which diagnosis traditionally depends on the manifestation of overtdisease symptoms. It will then be possible to identify the nucleic acidligands that are binding target molecules of relevance, and therebyidentify those target molecules. These embodiments will greatly expediteresearch into disease, and will provide many new target molecules thatcan be used in directed diagnostic and drug discovery programs.Furthermore, the nucleic acid ligands identified on the biochip thatbind to these target molecules may themselves have potential for use astherapeutic agents.

In the most preferred embodiment, the biochip contains both types ofnucleic acid ligands described in the previous two embodiments. Such abiochip will be able to detect or predict diseases where the diagnosticor prognostic criterion is (i) a change in the concentration ofmolecule(s) normally found in bodily fluid; and/or (ii) the presence ofa molecule not normally found in a bodily fluid of a healthy individual,e.g., a viral protein. FIG. 1 shows the use of such a biochip. For thesake of clarity, only a simple 4×4 array is illustrated; typicalembodiments may use arrays that are 100×100 or greater.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the use of a patterned array of nucleic acid ligands.Contacting the biochip with a test mixture results in a binding patternthat can be used to diagnose or predict disease.

FIG. 2 depicts a mechanism for detection in which an oligonucleotide,complementary in sequence to all or part of the nucleic acid ligand, isdisplaced from the nucleic acid ligand by binding of the targetmolecule.

FIG. 3 illustrates a mechanism for detection in which a nucleic acidligand is bound to the biochip via its interaction with anoligonucleotide, which oligonucleotide is covalently attached to thebiochip, and has a sequence complementary to all or part of the sequenceof the nucleic acid ligand.

FIG. 4 shows a mechanism for detection in which a small molecule isbound to the surface of the biochip, and the nucleic acid ligandsincorporate a binding site for this molecule.

FIG. 5 depicts a mechanism for detection of target molecule binding inwhich a nucleic acid ligand containing an additional binding site for asmall molecule is immobilized on a biochip.

FIG. 6 depicts a detection system that uses a cascade of nucleic acidhybridization.

DETAILED DESCRIPTION OF THE INVENTION

Contents

I. Glossary

II. Obtaining Nucleic Acid Ligands For Use on a Biochip

III. Fabrication of the Nucleic Acid Ligand Biochip

IV. Detection of Target Molecule Binding to Nucleic Acid Ligand UsingFluorescence Techniques

A. Generic Detection Techniques

B. Detection Using an Oligonucleotide with Sequence Complementary to theNucleic Acid Ligand

C. Incorporation of Small Molecule Binding Sites into Nucleic AcidLigands to Facilitate Detection of Target Molecule Binding

D. Detection Through a Hybridization Cascade

E. Direct Binding of Target Molecules to Spectroscopically DetectableNucleic Acid Ligands

F. Detection of Changes in Double-Helicity Accompanying Target Binding

G. Detection through the use of Interferometry

H. Detection of Covalently-Bound Target Molecules

V. Detection of Target Molecule Binding Through Methods That Do NotInvolve Fluorescence

A. Chemical Field Effect Transistors

B. Detection Through Surface Plasmon Resonance

C. Detection Through the Use of Mass Spectroscopy

D. Detection Through Atomic Force Microscopy (AFM) andScanning-Tunneling Microscopy (STM)

VI. Examples

I. Glossary

The following terms are intended to have the following general meaningsas they are used herein:

1. “SELEX” methodology refers to the selection of nucleic acid ligandswhich interact with a target in a desirable manner, for example bindingto a protein, with amplification of those selected nucleic acids asdescribed in detail above and in the SELEX patent applications.Iterative cycling of the selection/amplification steps allows selectionof one or a small number of nucleic acids which interact most stronglywith the target from a pool which contains a very large number ofnucleic acids. Cycling of the selection/amplification procedure iscontinued until a selected goal is achieved.

2. “SELEX target” or “target molecule” or “target” refers herein to anycompound upon which a nucleic acid can act in a predetermined desirablemanner. A SELEX target molecule can be a protein, peptide, nucleic acid,carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, pathogen, toxic substance, substrate,metabolite, transition state analog, cofactor, inhibitor, drug, dye,nutrient, growth factor, cell, tissue, etc., without limitation.Virtually any chemical or biological effector would be a suitable SELEXtarget. Molecules of any size can serve as SELEX targets. A target canalso be modified in certain ways to enhance the likelihood of aninteraction between the target and the nucleic acid.

3. “Tissue target” or “tissue” refers herein to a certain subset of theSELEX targets described above. According to this definition, tissues aremacromolecule in a heterogeneous environment. As used herein, tissuerefers to a single cell type, a collection of cell types, an aggregateof cells, or an aggregate of macromolecules. This differs from simplerSELEX targets which are typically isolated soluble molecules, such asproteins. In the preferred embodiment, tissues are insolublemacromolecules which are orders of magnitude larger than simpler SELEXtargets. Tissues are complex targets made up of numerous macromolecules,each macromolecule having numerous potential epitopes. The differentmacromolecules which comprise the numerous epitopes can be proteins,lipids, carbohydrates, etc., or combinations thereof. Tissues aregenerally a physical array of macromolecules that can be either fluid orrigid, both in terms of structure and composition. Extracellular matrixis an example of a more rigid tissue, both structurally andcompositionally, while a membrane bilayer is more fluid in structure andcomposition. Tissues are generally not soluble and remain in solidphase, and thus partitioning can be accomplished relatively easily.Tissue includes, but is not limited to, an aggregate of cells usually ofa particular kind together with their intercellular substance that formone of the structural materials commonly used to denote the generalcellular fabric of a given organ, e.g., kidney tissue, brain tissue. Thefour general classes of tissues are epithelial tissue, connectivetissue, nerve tissue and muscle tissue.

Examples of tissues which fall within this definition include, but arenot limited to, heterogeneous aggregates of macromolecule such as fibrinclots which are acellular; homogeneous or heterogeneous aggregates ofcells; higher ordered structures containing cells which have a specificfunction, such as organs, tumors, lymph nodes, arteries, etc.; andindividual cells. Tissues or cells can be in their natural environment,isolated, or in tissue culture. The tissue can be intact or modified.The modification can include numerous changes such as transformation,transfection, activation, and substructure isolation, e.g., cellmembranes, cell nuclei, cell organelles, etc.

Sources of the tissue, cell or subcellular structures can be obtainedfrom prokaryotes as well as eukaryotes. This includes human, animal,plant, bacterial, fungal and viral structures.

4. “Nucleic acid” means either DNA, RNA, single-stranded ordouble-stranded and any chemical modifications thereof. Modificationsinclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, and fluxionality to the individualnucleic acid bases or to the nucleic acid as a whole. Such modificationsinclude, but are not limited to, modified bases such as 2′-positionsugar modifications, 5-position pyrimidine modifications, 8-positionpurine modifications, modifications at cytosine exocyclic amines,substitution of 5-bromo-uracil; backbone modifications, methylations,unusual base-pairing combinations such as the isobases isocytidine andisoguanidine and the like. Modifications can also include 3′ and 5′modifications such as capping. Modifications that occur after each roundof amplification are also compatible with this invention.Post-amplification modifications can be reversibly or irreversibly addedafter each round of amplification. Virtually any modification of thenucleic acid is contemplated by this invention.

5. “Nucleic acid test mixture” or “nucleic acid candidate mixture”refers herein to a mixture of nucleic acids of differing, randomizedsequence. The source of a “nucleic acid test mixture” can be fromnaturally-occurring nucleic acids or fragments thereof, chemicallysynthesized nucleic acids, enzymatically synthesized nucleic acids ornucleic acids made by a combination of the foregoing techniques. In apreferred embodiment, each nucleic acid has fixed sequences surroundinga randomized region to facilitate the amplification process. The lengthof the randomized section of the nucleic acid is generally between 8 and250 nucleotides, preferably between 8 and 60 nucleotides.

6. “Nucleic acid ligand” refers herein to a nucleic acid which has beenisolated from the nucleic acid candidate mixture that acts on a targetin a desirable manner. Examples of actions on a target in a desirablemanner include, but are not limited to binding of the target,catalytically changing the target, reacting with the target in a waywhich modifies/alters the target or the functional activity of thetarget, covalently attaching to the target as in a suicide inhibitor,facilitating the reaction between the target and another molecule. Inmost, but not all, instances this desirable manner is binding to thetarget. In the most preferred embodiment, a nucleic acid ligand is anon-naturally occurring nucleic acid sequence having a specific bindingaffinity for a target molecule, such target molecule being a threedimensional chemical structure other than a polynucleotide that binds tosaid nucleic acid ligand through a mechanism which predominantly dependson Watson/Crick base pairing or triple helix binding, wherein saidnucleic acid ligand is not a nucleic acid having the known physiologicalfunction of being bound by the target molecule. Nucleic acid ligandincludes nucleic acid sequences that are substantially homologous to thenucleic acid ligands actually isolated by the SELEX procedures. Bysubstantially homologous it is meant a degree of primary sequencehomology in excess of 70%, most preferably in excess of 80%. In the pastit has been shown that various nucleic acid ligands to a specific targetwith little or no primary homology may have substantially the sameability to bind the target. For these reasons, this invention alsoincludes nucleic acid ligands that have substantially the same abilityto bind a target as the nucleic acid ligands identified by the SELEXprocess. Substantially the same ability to bind a target means that theaffinity is within a few orders of magnitude of the affinity of theligands described herein. It is well within the skill of those ofordinary skill in the art to determine whether a givensequence—substantially homologous to those specifically describedherein—has substantially the same ability to bind a target.

7. “Bodily fluid” refers herein to a mixture of macromolecules obtainedfrom an organism. This includes, but is not limited to, blood plasma,urine, semen, saliva, lymph fluid, meningial fluid, amniotic fluid,glandular fluid, and cerebrospinal fluid. This also includesexperimentally separated fractions of all of the preceding. “Bodilyfluid” also includes solutions or mixtures containing homogenized solidmaterial, such as feces, tissues, and biopsy samples.

8. “Test mixture” refers herein to any sample that contains a pluralityof molecules, the identity of at least some of which can be detectedusing a nucleic acid ligand biochip. This includes, but is not limitedto, bodily fluids as defined above, and any sample for environmental andtoxicology testing such as contaminated water and industrial effluent.

9. “Biochip” is any microfabricated solid surface to which molecules maybe attached through either covalent or non-covalent bonds. Thisincludes, but is not limited to, Langmuir-Bodgett films. functionalizedglass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold,and silver. Any other material known in the art that is capable ofhaving functional groups such as amino, carboxyl, thiol or hydroxylincorporated on its surface, is contemplated. This includes planarsurfaces, and also spherical surfaces.

II. Obtaining Nucleic Acid Ligands for Use on a Biochip

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. Pat. application Ser. No. 08/123,935,filed Sep. 17, 1993, abandoned in favor of U.S. patent application Ser.No. 08/443,959, filed May 18, 1995, now abandoned, entitled“Photoselection of Nucleic Acid Ligands,” describes a SELEX based methodfor selecting nucleic acid ligands containing photoreactive groupscapable of binding and/or photocrosslinking to and/or photoinactivatinga target molecule. U.S. patent application Ser. No. 08/134,028, filedOct. 7, 1993, abandoned in favor of U.S. patent application Ser. No.08/443,957, filed May 18, 1995, now U.S. Pat. No. 5,580,737, entitled“High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, termed Counter-SELEX. U.S. patent application Ser.No. 08/143,564, filed Oct. 25, 1993, abandoned in favor of U.S. patentapplication Ser. No. 08/461,069, filed Jun. 5, 1995, now U.S. Pat. No.5,567,588, entitled “Systematic Evolution of Ligands by EXponentialEnrichment: Solution SELEX,” describes a SELEX-based method whichachieves highly efficient partitioning between oligonucleotide havinghigh and low affinity for a target molecule. U.S. patent applicationSer. No. 08/442,062, filed May 16 1995, entitled “Methods of ProducingNucleic Acid Ligands,” now U.S. Pat. No. 5,595,877, describes methodsfor obtaining improved nucleic acid ligands after SELEX has beenperformed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8,1995, now U.S. Pat. No. 5,705,337, entitled “Systematic Evolution ofLigands by EXponential Enrichment: Chemi-SELEX,” describes methods forcovalently linking a ligand to its target. Of particular note to theinstant application, U.S. patent application Ser. No. 08/434,425, filedMay 3, 1995, now U.S. Pat. No. 5,789,157, entitled “Tissue SELEX,”describes methods for identifying and preparing nucleic acid ligandsagainst an entire tissue, wherein tissue is defined as a single celltype, a collection of cell types, an aggregate of cells or an aggregateof macromolecules. Examples of candidate tissues include tumors andblood plasma. These methods are also disclosed in great detail in U.S.patent application Ser. Nos. 08/434,425, now U.S. Pat. No. 5,789,157,08/437,667, now U.S. Pat. No. 5,864,026, 08/434,001, now U.S. Pat. No.5,712,375, 08/433,585, now U.S. Pat. No. 5,763,566, 08/906,955, now U.S.Pat. No. 6,013,443, 08/433,124, now U.S. Pat. No. 5,750,342, and08/433,126, now U.S. Pat. No. 5,688,935.

The SELEX method encompasses combining selected oligonucleotide withother selected oligonucleotide and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284,063, filed Aug.2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459, and U.S.patent application Ser. No. 08/234,997, filed Apr. 28, 1994, now U.S.Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands byExponential Enrichment: Blended SELEX,” respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties ofoligonucleotide with the desirable properties of other molecules. Eachof the above described patent applications which describe modificationsof the basic SELEX procedure are specifically incorporated by referenceherein in their entirety. Any variation of the SELEX method that allowsfor the incorporation of a detectable group (such as a fluorescentchemical, or a group such as digoxigenin that is recognized by anantibody), an affinity group (such as biotin), a reactive group (such asa photoreactive group or a chemical group that permits attachment of theligand to a biochip surface), or of the binding site sequence of anotherSELEX target is also contemplated in the subject invention.

In a preferred embodiment, the SELEX procedure is carried out in thepresence of blood plasma in order ultimately to obtain nucleic acidligands that bind the target molecule specifically without thepossibility that they cross react with other molecules in plasma. Someexamples of nucleic acid ligands that have utility in the biochipinclude, but are not limited to, ligands against HIV-1 tat protein (U.S.Pat. Nos. 5,527,894 and 5,637,461), HIV-1 gag (U.s. Pat. No. 5,726,017),HIV-1 integrase (U.S. Pat. No. 5,587,468), HIV-1 nucleocapsid components(U.S. Pat. Nos. 5,654,151 and 5,635,615) HIV-1 reverse transcriptase(U.S. Pat. Nos. 5,496,938 and 5,503,978), thrombin (U.S. Pat. Nos.5,476,766 and 5,543,293), basic fibroblast growth factor (U.S. Pat. Nos.5,459,015 and 5,639,868), vascular endothelial growth factor (U.S. Pat.No. 5,811,533), insulin receptor antibodies (U.S. patent applicationSer. No. 08/248,632, now abandoned), the tachykinin substance P (U.S.Pat. No. 5,648,214 and 5,637,682), immunoglobulin E (U.S. Pat. Nos.5,629,155 and 5,686,592), secretory phospholipase A₂ (U.S. Pat. No.5,622,828), TGFβ (U.S. Pat. Nos. 5,731,144 and 5,731,424), plateletderived growth factor (U.S. Pat. No. 5,668,264, U.S. Pat. No. 5,723,594and World Patent No. WO 96/38579), human kerotinocyte growth factor(World Pat. No. WO 96/38579, U.S. Pat. No. 5,846,713), chorionicgonadotropin (U.S. Pat. Nos. 5,837,456 and 5,849,890), lectins (U.S.Pat. No. 5,780,228 and U.S. patent application Ser. Nos. 08/477,829, nowabandoned, and 08/472,256, now U.S. Pat. No. 6,001,988), cytokines (U.S.patent application Ser. Nos. 08/477,527, now U.S. Pat. No. 5,972,599,and 08/481,710, now U.S. Pat. No. 6,028,186), lupus antibodies (U.S.patent application Ser. No. 08/520,932, now abandoned), and complementsystem proteins (U.S. patent application Ser. No. 08/595,335, nowabandoned).

As described in the SELEX patent applications, it is possible to createnucleic acid ligands with constant and random sequence regions. In aparticularly preferred embodiment, nucleic acid ligands will besynthesized that have a common short sequence (seq. A) located at apredetermined position. The initial candidate mixture of nucleic acidswill then be contacted with a solid support, preferably a column,containing an immobilized nucleic acid (seq. A′) complementary insequence to the common short sequence on each ligand. The pool ofligands will then bind to the column through complementary base pairingbetween A and A′. A mixture containing the target molecule(s) will thenbe passed over the column, and ligands that are displaced from thecolumn will be collected. The displacement of these ligands indicatesthat the binding of the target molecule alters the conformation of theligand in such a manner that the common short sequence is no longer ableto bind to its complementary sequence. In a related embodiment, theinitial candidate mixture of nucleic acid ligands will be contacted withthe target molecule, and binding will be allowed to occur in solutionphase. The nucleic acid ligands will then be contacted with the columndescribed above. Nucleic acid ligands that have bound target in such away that sequence A is not able to hybridize to column-bound sequence A′will pass through the column, and can be collected. The nucleic acidligands obtained in these two embodiments will be used in the biochip asdescribed in detail below in the section entitled “Detection of TargetMolecule Binding to Nucleic Acid Ligand Using Fluorescence Techniques.”

One of the most powerful aspects of the present invention is the abilityto identify an extremely large number of nucleic acid ligands thatrecognize correspondingly large numbers of targets in a biologicalsample. In many embodiments of the invention, the larger the number oftargets that are identifiable in a solution or mixture the better. TheSELEX process allows for the selection of nucleic acid ligands withoutknowing what molecular target they bind to. For diagnostic andprognostic purposes, the specific targets can be almost irrelevant aslong as some pattern of target presence is indicative of a certaincondition. By this process, it is likely that the presence of multipletargets, seemingly unrelated, would be determined to be diagnostic orprognostic of a given condition. This invention frees the investigatorfrom having to determine which targets to detect within a givenbiological sample. Because SELEX can simultaneously identify ligands tohuge numbers of epitopes within a complex sample, a new diagnosticapproach can be employed that does not rely on a previous knowledge ofwhich targets are critical.

Thus, in certain aspects of the invention, the biochip may be comprisedof literally thousands of nucleic acid ligands to indeterminate targets.In other embodiments, the targets for each attached nucleic acid ligandmay be either predetermined based on the nature of the SELEX protocolemployed, or determined after the nucleic acid ligand was identified.

III. Production of the Nucleic Acid Ligand Biochip

The production of biochips on which nucleic acids are immobilized iswell known in the art. The biochip may be a Langmuir-Bodgett film,functionalized glass, germanium, silicon, PTFE, polystyrene, galliumarsenide, gold, silver, membrane, nylon, PVP, or any other materialknown in the art that is capable of having functional groups such asamino, carboxyl, Diels-Alder reactants, thiol or hydroxyl incorporatedon its surface. Preferably, these groups are then covalently attached tocrosslinking agents, so that the subsequent attachment of the nucleicacid ligands and their interaction with target molecules will occur insolution without hindrance from the biochip. Typical crosslinking groupsinclude ethylene glycol oligomer, diamines, and amino acids. Anysuitable technique useful for immobilizing a nucleic acid ligand to abiochip is contemplated by this invention.

In one embodiment, one or more nucleic acid ligands will be attached tothe support by photolithography using a photoreactive protecting groupon a coupling agent. Such a technique is disclosed in McGall et al.,U.S. Pat. No. 5,412,087. Thiolpropionate having a photochemicallyremovable protecting group is covalently coupled to functional groups onthe surface of the biochip. Light of the appropriate wavelength is thenused to illuminate predefined regions of the surface, resulting inphotodeprotection of the thiol group. A mask will be used to ensure thatphotodeprotection only takes place at the desired sites or addresses.Nucleic acid ligands containing thiol reactive groups, such asmaleimides, are then attached to the deprotected regions. The unboundnucleic acid ligand will then be washed away, and the process repeatedat another location with another nucleic acid ligand. A similar methoduses a 5′-nitroveratryl protected thymidine linked to an aminatedbiochip via a linkage to the 3′-hydroxyl group (Fodor et al. (1991)Science 251:767-773). Photodeprotection of the thymidine derivativeallows a phosphoramidite activated monomer (or oligomer) to react atthis site. Other methods use a photoactivatable biotin derivative tospatially localize avidin binding. The avidin, by virtue of its abilityto bind more than one biotin group at a time, will in turn be used as ameans for spatially localizing a biotin-linked nucleic acid ligand tothe biochip (Barrett et al., U.S. Pat. No. 5,252,743 and PCT 91/07807).In principle, the photodeprotection of a caged binding agent could beused for any ligand-receptor pair where one member of the pair is asmall molecule capable of being protected by a photolabile group. Otherexamples of such ligand-receptor pairing include mannose andconcanavalin A, cyclic AMP and anti-cAMP antibodies, andtetrahydrofolate and folate binding proteins (U.S. Pat. No. 5,252,743).

In another embodiment, the regions of the biochip that come into contactwith the nucleic acid ligand at each attachment photoactivation step arespatially restricted. This may be done by placing the support on a blockcontaining channels through which the nucleic acid ligand will bepumped, with each channel giving the nucleic acid ligand access to onlya small region of the biochip. This prevents accidental binding ofnucleic acid ligand to non-photoactivated regions. Furthermore, it willbe used to permit the simultaneous attachment of several differentnucleic acid ligands to the support. In this embodiment, a mask allowsfor the patterned illumination and consequent photoactivation of severalregions of the biochip at the same time. If the area surrounding eachphotoactivated region is segregated from the neighboring region by theaforementioned channel, then different nucleic acid ligands will bedelivered to these photoactivated regions by pumping each nucleic acidligand through a different channel (Winkler et al., U.S. Pat. No.5,384,261).

The photoactivated regions in the methods described above will be atleast as small as 50 mm². It has been shown that >250,000 binding sitesper square centimeter is easily achievable with visible light; the upperlimit is determined only by the diffraction limit of light (Fodor el al.(1991) Science 251:767-773). Therefore, photoactivation usingelectromagnetic radiation of a shorter wavelength will be used togenerate correspondingly denser binding arrays. If the biochip istransparent to the incident radiation it will be possible tosimultaneously perform this process on a vertical stack of biochips,greatly increasing the efficiency of biochip production.

Alternatively, some form of template-stamping is contemplated, wherein atemplate containing the ordered array of nucleic acid ligands (andpossibly manufactured as described above) will be used to deposit thesame ordered array on multiple biochips.

In a further embodiment, the nucleic acid ligand array will be formed onthe biochip by an “inkjet” method, whereby the ligands are deposited byelectromechanical dispensers at defined locations. An ink-jet dispensercapable of forming arrays of probes with a density approaching onethousand per square centimeter is described in Hayes et al., U.S. Pat.No. 5,658,802.

IV. Detection of Target Molecule Binding to Nucleic Acid Ligand UsingFluorescence Techniques

A. Generic Detection Techniques

In one embodiment, protein target molecules bound to nucleic acidligands on the surface of the biochip will be detected by the additionof chemicals that non-specifically bind to all proteins but not tonucleic acids. More generally, such agents bind to proteins preferablyover nucleic acids. Any fluorescent chemical that is known in the art tobind proteins non-specifically will be suitable. Suitable examplesinclude the dyes Nanorange and Cytoprobe, available from MolecularProbes, Inc.

In a preferred embodiment, specific detection of protein that iscovalently (or noncovalently) coupled to immobilized aptamer can beachieved by taking advantage of the different reactivities of nucleicacid and protein functional groups. Nucleic acids have no strongnucleophiles, whereas lysine and cysteine side-chains provide activenucleophiles to proteins. Lysine is a moderately abundant amino acid,comprising 4-6% of the side chains of most proteins. Cysteine variesconsiderably more in its abundance, and is often sequestered indisulfide bonds with other cysteine residues, rendering it lessavailable for reaction.

Accordingly, the chemistry of protein modification through lysineresidues is well-developed. A large number of fluorophores or othertagging agents have been developed which react with lysine. The mostcommon chemistries rely on the reaction of lysine withhydroxysuccinamide (NHS) esters, isothiocyanates, or in a variety ofaldehyde reactions.

In another embodiment, target molecules bound to nucleic acid ligandswill be detected on the biochip surface through the use of a sandwichassay. This method is well known to those skilled in the art. A sandwichassay uses antibodies that recognize specific bound target molecules,preferably binding at a site distinct from that recognized by thenucleic acid ligand. In such sandwich assays, the antibodies may befluorescently labeled, or the bound antibodies may themselves bedetected by contacting the biochip with fluorescently labeled protein A,which binds all immunoglobulins. Alternatively, secondary antibodiesspecific for the immunoglobulin subtype of the first (primary) antibodywill be contacted with the biochip. The secondary antibodies may befluorescently labeled, or they may be conjugated to a reporter enzyme,which enzyme catalyses the production of a detectable compound. Sandwichassays have the potential to greatly amplify the detectable signal, inthis case by the ability of the secondary antibody to bind to multiplesites on the primary antibody. All variations of the sandwich assayknown in the art are contemplated in the subject invention.

In a related sandwich assay embodiment, the bound target molecule willbe detected by the use of a second nucleic acid ligand, which binds to asite on the bound target distinct from that recognized by thebiochip-bound nucleic acid ligand. As described in the paragraph above,the second nucleic acid ligand may be fluorescently labeled, or it maybe conjugated to biotin, allowing fluorescently labeled avidin, or anavidin conjugated reporter enzyme, to then bind to the bound secondnucleic acid ligand. Alternatively, the first and second nucleic acidligands may be labeled in an appropriate manner so that they form anenergy transfer pair, as described below in the section entitled“Detection Using an Oligonucleotide with Sequence Complementary to theNucleic Acid Ligand.”

In another embodiment, target molecule binding will be detected using acompetition assay, well known to those skilled in the art. Followingcontacting of the biochip-bound nucleic acid ligands with the testmixture, a solution containing a predetermined amount of each target forwhich binding data is sought is added. These target molecules arefluorescently labeled by any of the ways known in the art in order topermit their detection. The labeled target molecules compete for bindingto the immobilized nucleic acid ligand. An equilibrium will beestablished, and the amount of labeled molecule bound at each site willbe used to calculate the amount of each target molecule contained withinthe original test mixture.

In another embodiment, protein enzymes bound to aptamers can be detectedby an assay of enzyme activity.

B. Detection Using an Oligonucleotide with Sequence Complementary to theNucleic Acid Ligand

In certain preferred embodiments (FIG. 2), nucleic acid ligands (21)containing a constant sequence associated with the binding site for thetarget molecule will be localized to specific regions of a biochip (22).The synthesis of such nucleic acid ligands is described above in thesection entitled “Obtaining Nucleic Acid Ligands For Use on a Biochip”.The biochip-bound nucleic acid ligands will then be hybridized with anoligonucleotide (23) complementary in sequence to the constant region.Contacting this biochip with a test mixture will lead to displacement(24) of oligonucleotide from nucleic acid ligands that bind to theirtarget molecule (25). In a further embodiment (FIG. 3), a biochip (31)will be synthesized upon which the complementary oligonucleotide (32) isimmobilized by any of the methods known in the art. The nucleic acidligands (33) will then be deposited at specific locations on thebiochip, whereupon they will become associated with the oligonucleotideby base pairing. The biochip will then be contacted with the testmixture. Target molecule binding (34) will lead to the disruption ofbase pairing between the nucleic acid ligand and the support boundoligonucleotide (35), and hence displacement of the nucleic acid ligandfrom the biochip will occur.

In the preceding embodiments, the displaced nucleic acid is labeled (26,36) with fluorescein, tetramethylrhodamine. Texas Red, or any otherfluorescent molecule known in the art, leading to a decrease influorescence intensity at the site of target molecule binding. The levelof label detected at each address on the biochip will then varyinversely with the amount of target molecule in the mixture beingassayed. Alternatively, the nucleic acid ligand and the oligonucleotideconstitute an energy transfer pair. For example, one member of the pairwill be labeled with tetramethylrhodamine and the other will befluorescein labeled. The fluorescein-based fluorescence of such acomplex is quenched when illuminated with blue light, as the green lightemitted by the fluorescein will be absorbed by the tetramethylrhodaminegroup; the rhodamine-based fluorescence of this complex is not quenched.Separation of the two halves of this energy transfer pair occurs upontarget molecule binding, and leads to a change in the emission profileat such sites on the biochip. The displacement of thetetramethylrhodamine labeled molecule will lead to the sudden appearanceof fluorescein-based fluorescence at this site on the biochip, with theconcomitant loss of rhodamine-based fluorescence. The simultaneouschange in two different emission profiles will enable ratiometricimaging of each site to be performed, allowing sensitive measurement oftarget molecule binding. It is clear to those skilled in the art thatany energy transfer pair can be used in this embodiment, providing thatthey have appropriately matched excitation and emission spectra.

In an alternative embodiment, the displaced nucleic acid is conjugatedto one member of an affinity pair, such as biotin. A detectable moleculeis then conjugated to the other member of the affinity pair, for exampleavidin. After the test mixture is applied to the biochip, the conjugateddetectable molecule is added. The amount of detectable molecule at eachsite on the biochip will vary inversely with the amount of targetmolecule present in the test mixture. In another embodiment, thedisplaced nucleic acid will be biotin labeled, and can be detected byaddition of fluorescently labeled avidin; the avidin itself will then belinked to another fluorescently labeled, biotin-conjugated compound. Thebiotin group on the displaced oligonucleotide can also be used to bindan avidin-linked reporter enzyme; the enzyme will then catalyze areaction leading to the deposition of a detectable compound.Alternatively, the reporter enzyme will catalyze the production of aninsoluble product that will locally quench the fluorescence of anintrinsically-fluorescent biochip. In another embodiment of thedisplacement assay, the displaced oligonucleotide will be labeled withan immunologically-detectable probe, such as digoxigenin. The displacedoligonucleotide will then be bound by a first set of antibodies thatspecifically recognize the probe. These first antibodies will then berecognized and bound by a second set of antibodies that arefluorescently labeled or conjugated to a reporter enzyme. Manyvariations on these examples are well known to those skilled in the art.

In variations of the preceding embodiments, the nucleic acid ligand willnot contain a constant sequence region as described above. In theseembodiments, the oligonucleotide will have a sequence that iscomplementary to all, or part of, the nucleic acid ligand. Thus, eachnucleic acid ligand will bind an oligonucleotide with a unique sequence.The oligonucleotides can be displaced from biochip-localized nucleicacid ligands as described above upon target binding. Alternatively, theoligonucleotides will be localized to specific locations on the biochipas described above, which will in turn result in the specificlocalization of nucleic acid ligands by complementary base-pairing tothe oligonucleotides. Target molecule binding will displace the nucleicacid ligand from the biochip in this case, as described above. In eachcase, the oligonucleotide and/or the nucleic acid ligand will be labeledas described above.

In other embodiments, nucleic acid ligands will be localized to specificregions of the biochip. Following contacting with the test mixture, thebiochip will then be contacted with a solution containing either (i)oligonucleotide with sequence complementary to the constant region ofthe nucleic acid ligand; or (ii) oligonucleotides with sequencecomplementary to all or part of each nucleic acid ligand, which liganddoes not contain a constant sequence region as described above in thissection. In these cases, binding of target will prevent the subsequentbinding of oligonucleotide. Again, the oligonucleotide and the nucleicacid ligand can be labeled as described above in this section to monitorthe binding of oligonucleotide.

C. Incorporation of Small Molecule Binding Sites into Nucleic AcidLigands to Facilitate Target Molecule Binding

In another embodiment, SELEX will be performed using a pool of nucleicacids containing a binding site for a particular small molecule. Anexample of such a small molecule is the caffeine analogue theophylline.Single stranded nucleic acid ligands against this molecule form adouble-stranded stem with a hairpin loop in which the 5′ and 3′ ends ofthe molecule are close to one another. This structure only forms in thepresence of theophylline. In this embodiment, a candidate mixture oftheophylline ligands will be synthesized with random sequence in thehairpin loop region, and the candidates will then be passed over a solidsupport, preferably a column, to which theophylline has been attached.The candidate ligands will bind tightly to theophylline, and will becomeimmobilized on the column. The mixture containing target molecules willbe added to the column, and ligands that are eluted will be collected.This will select for ligands that bind their target molecules in such away that the ligand will no longer bind to theophylline. Such ligandswill be displaced because the adoption of the structure that binds thetarget molecule will disrupt the structure that binds the theophylline.The ligands will be refined in the standard ways described in the SELEXpatent applications. A biochip (FIG. 4) will then be fabricated (41) onwhich theophylline (42) is attached by any of the methods known in theart. One or more individual species of the nucleic acid ligands (43)will then be attached at defined locations on the biochip, where theybind tightly to the theophylline. Contacting of the test mixture withthe biochip leads to the displacement (44) from the biochip of nucleicacid ligands that bind to their cognate target molecule (45). Thedisplacement will be detected by any of the means detailed above (46).This technique will be used with any nucleic acid ligand that forms ahairpin-type structure similar to theophylline, or any other nucleicacid ligand that can be synthesized with additional random sequence, andwill then bind to two different compounds in a mutually exclusivemanner, such that the binding of one compound will displace the other.

In a related embodiment (FIG. 5), nucleic acid ligands containing abinding site for a particular small molecule, such as theophylline, anda randomized segment will be synthesized as described in the aboveparagraph. The ligands (51) will also be labeled with both members of anenergy transfer pair (52, 53), such that in the presence of the smallmolecule, these groups are close to one another, and fluorescence isquenched. The ligands will then be deposited at specific regions of thebiochip (54), and the small molecule (55) will be added to the biochip.The ligands will adopt the structure that binds the small molecule, andfluorescence will be quenched. The test mixture will then be added tothe biochip, and target molecules (56) will displace the small moleculefrom the appropriate nucleic acid ligands. In order to bind the targetmolecules, the nucleic acid ligands will undergo a conformational changein which the two halves of the energy transfer pair are no longer nextto one another (57). This will result in a change in the fluorescenceprofile at each site on the biochip where a target molecule has beenbound. This embodiment will also be used for ligands that contain abinding for any small molecule, provided that such ligands undergo aconformational change upon displacement of the small molecule by thetarget molecule.

A different displacement scheme will use the target-molecule-dependentdisplacement of a labeled single-stranded DNA-binding protein from asupport bound nucleic acid ligand.

McGall el al., supra, suggest a technique for simultaneously identifyingmultiple target nucleic acid sequences using multiple probes. In themethod contemplated, a first set of labeled probes against specifictargets is synthesized, with each probe containing an additionalsequence that is unique for that particular probe. These uniquesequences are complementary to a second set of oligonucleotidesimmobilized on a biochip. The authors envision contacting the target andthe first set of probes in solution, then adding the complexes formed tothe biochip. The additional unique sequence region of each probe willlocalize that complex to a specific address on the biochip via itsinteraction with the second probe bound at that site. Because there aremethods known in the art that can be used to partition bound nucleicacid ligand from unbound, this technique can be applied to the instantinvention. Specifically, nucleic acid ligands will be synthesized withan additional sequence that will be different for each species ofnucleic acid ligand and will preferably be distant from the residuesimportant for the specific binding interaction. The biochip will containoligonucleotides with sequence complementary to the unique region ofeach nucleic acid ligand species. Each nucleic acid ligand will alsohave a detectable group, such as a fluorophore and/or a means forlinking the nucleic acid ligand to another detectable molecule asdescribed above. Alternatively, the second set of biochip-localizednucleic acids and the nucleic acid ligands themselves can be labeled insuch a way that they form an energy transfer pair, as described above.

D. Detection of Binding Through a Signal-Amplifying HybridizationCascade

Another series of embodiments of the instant invention will involve theuse of a set of mutually-complementary nucleic acids. In all themethods, the nucleic acid ligand binds to a target molecule whereuponthe nucleic acid ligand undergoes a conformational change that allowsother nucleic acids to hybridize thereto. The nucleic acids thathybridize to the target-bound nucleic acid ligand also undergo aconformational change during hybridization that similarly allows othernucleic acids to hybridize thereto. This chain reaction ofconformational change and hybridization will continue, forming anincreasingly large intermolecular hybridization complex at each site onthe biochip where a nucleic acid ligand binds to a target molecule. Anynucleic acid structure that undergoes a hybridization-promotingconformational change upon (i) binding to a target molecule, and/or (ii)hybridizing to another nucleic acid, is suitable for use in thisembodiment. The use of cascade hybridization is described in U. S.patent application Ser. No. 60/068,135, entitled “System for AmplifyingFluorescent Signal Through Hybridization Cascade,” filed Dec. 15, 1997,now abandoned, and specifically incorporated herein by reference.

The hybridizing nucleic acids will be labeled with a fluorescent groupand a quenching group at positions that are spatially adjacent only whenthe nucleic acid is not participating in a hybridization reaction.Therefore, the formation of the intermolecular complex will beaccompanied by the generation of an increasing large fluorescent signalat each site on the biochip where a target molecule binds to a nucleicacid ligand. This signal can be detected by any of the fluorescencetechniques known in the art.

In a preferred embodiment, a set of mutually-complementary stem-loopnucleic acids will be synthesized. A nucleic acid ligand will bedesigned with a stem-loop structure, in which the target moleculebinding site is located in the loop region. Each said species of nucleicacid ligand will be immobilized at discrete locations on the biochip.The stem region will comprise two partially complementary “arms” ofsequence A and B (FIG. 6) that can undergo limited pairing to form animperfect intramolecular double helix (61). This nucleic acid ligandwill undergo a structural change upon target molecule binding such thatthe stem region is completely disrupted (62). Three or more further setsof imperfect stem-loop nucleic acids will also be synthesized. The firstfurther set will be identical to the biochip-bound nucleic acid ligand,but will not contain the target molecule binding site in the loop region(63). The sequences of the stem regions of the latter two sets arerepresented as C′/A′ (64) and B′/C (65), and are chosen so that they canbind perfectly to (i) one of the arms of the nucleic acid ligand stem(A′ pairs perfectly with A, and B′ pairs perfectly with B), and (ii) thearms of the second set can bind perfectly to the arms of the third set(C′ pairs perfectly with C). The three sets will further comprise afluorescent group (66) and a quenching group (67) located at positionsthat are spatially adjacent only when the imperfect stem structure isformed. A biochip with the stem-loop nucleic acid ligands will becontacted with a test mixture, and target molecule binding will lead tothe disruption of the stem region of said nucleic acid ligands. Bothsequences A and B will be available for base-pairing. The biochip willthen be contacted with a solution of all three sets of nucleic acids.The arms of the stems of these latter nucleic acids will then hybridizeto any nucleic acid ligand that has undergone a target-binding reaction(68). Upon binding to the nucleic acid ligand arms, the stem regions ofthe second and third set of nucleic acids will be similarly disrupted,and the unhybridized arms can then hybridize to their complementarysequences. This process is driven by the favorable free energydifference between imperfect and the perfect double helices, and willcontinue until one of the nucleic acids is depleted from the solutionphase. At each hybridization step, another arm sequence becomesavailable for complementary base pairing, leading to the ultimateformation of a multimolecular complex of intermolecular double helices.Each hybridization step is accompanied by the spatial separation of thequenching group from the fluorescent group, resulting in a highlyfluorescent signal (69) being generated at the site on the biochip wherea single target molecule originally bound to a single nucleic acidligand. In this embodiment, the original fluorescent signal is highlyamplified by the cascade of hybridization.

E. Direct Binding of Target Molecules to Spectroscopically DetectableNucleic Acid Ligands

In another embodiment, one or more spectroscopically detectable labelednucleic acid ligands will be immobilized on biochips. The synthesis ofsuch ligands is disclosed in Pittner et al., U.S. Pat. No. 5,641,629 andU.S. Pat. No. 5,650,275, both of which are specifically incorporatedherein by reference. The labels on such ligands undergo a detectablechange in fluorescence intensity, fluorescence polarization orfluorescence lifetime upon binding of the nucleic acid ligand to itstarget molecule. Suitable labels include fluorescent labels (e.g.fluorescein, Texas Red), luminescent labels (e.g. luciferin, acridiniumesters), energy transfer labels (e.g. fluorescein andtetramethylrhodamine), and near IR labels (e.g. dicyanines, La JollaBlue dye). Binding of the target molecule to the labeled ligand will bedetected by measuring any change in fluorescence. These include, but arenot limited to, changes in fluorescence polarization, fluorescenceanisotropy, fluorescence intensity, and fluorescence lifetime. Thesemeasurements will be made continuously, or in a dynamic manner.Locations on the biochip where a difference is detectable will then beknown to have bound target molecules, allowing the quantification ofeach target molecule in the test mixture.

In a preferred embodiment, nucleic acid ligands bound to the biochipwill be labeled with one or more phosphorescent groups. These groupswill be incorporated into the nucleic acid ligand such that at leastsome of them are within the binding site of the target molecule. Thephosphorescent groups will be chosen from those known in the art so thatthey have an emission half life which is longer by a predeterminedamount than the half life of non-specific binding of an inappropriatetarget molecule to a nucleic acid ligand. The medium in which thebiochip is incubated will contain a predetermined amount of a quenchingagent that can effectively quench the phosphorescence. When targetmolecule binds to a nucleic acid ligand in a specific manner, then thephosphorescent groups will be protected from the quenching agents. Ifthe biochip is then illuminated with light of the appropriatewavelength, the phosphorescent groups of nucleic acid ligands withspecifically bound target molecules will phosphoresce, and hence lightwill detected at sites on the biochip where ligand is bound. Thephosphorescence of nucleic acid ligands that are unbound will bequenched, and so no light will be detected at such sites on the biochip.The phosphorescence of nucleic acid ligands that bind an inappropriate,non-cognate target molecule will also be quenched, as the half life forthe formation of these complexes will be much shorter than the emissionhalf life of the phosphorescence groups. In other words, the individualphosphorescent groups in a non-specific complex will be many more timeslikely to encounter a quenching group in the solvent prior to photonemission than will those same groups in a specific complex. Any suitablephosphorescent group with an emission half life greater than the halflife of formation of a specific nucleic acid ligand-target complex iscontemplated in the present invention. If the detection ofphosphorescence is delayed by a predetermined amount of time followingexcitation illumination, then the phosphorescence signal can bedistinguished from the background fluorescence signals, as these lattersignals have a much shorter emission half life. This delay will alsofurther enhance the specificity of detection, as only truly tightlybound nucleic acid ligands will be protected from quenching.Furthermore, a series of phosphorescence images of the biochip will beobtained, with optional brief washing of the biochip between eachexposure; the resulting series of images will then be integrated. Thiswill enable non-specific signal to be further distinguished fromnon-specific signal, as the specific binding will persist betweenexposures, whereas the non-specific binding will not.

In another embodiment, the technique described in the precedingparagraph will be carried out using fluorescence groups on the nucleicacid ligands, rather than phosphorescence groups.

F. Detection of Changes in Double-Helicity Accompanying Target Binding

In another embodiment, target molecule binding will be assessed bymonitoring changes in the degree of double-strandedness of each nucleicacid ligand. It is known that nucleic acid ligands undergo structuralchanges upon binding to target, such as the formation, or expansion, ofdouble stranded regions. In the instant invention, these changes will bedetected by adding a fluorescent intercalating dye, such as ethidiumbromide, to the biochip, and measuring fluorescence levels at eachlocation on the biochip in the presence and absence of the test mixture.A similar technique is suggested in Lockhart et al. (U.S. Pat. No.5,556,752) for determining oligonucleotide probe hybridization to atarget nucleic acid sequence.

G. Detection Through the use of Interferometry

In another embodiment, target molecule binding will be detected by theuse of an interferometric sensing system. A suitable system is describedin Lin et al. (1997) Science 278: 840-842. Nucleic acid ligands will beattached to a microporous biochip surface. Illumination of this surfacewith white light produces an interference pattern. This results fromlight being reflected from the top and bottom of the porous biochipsurface. Interaction of a target molecule with a nucleic acid ligandlocally alters the refractive index of the biochip surface, and this inturn locally alters the wavelength of the interference fringe pattern.This can be measured by, for example, a charge coupled device camera.

H. Detection of Covalently Bound Target Molecules

In another embodiment, methods known in the art that allow for thesynthesis of nucleic acid ligands containing one or more photoreactivegroups, such as iodouridine, will be used. These ligands are capable ofbinding to their target, and then becoming covalently attached to thetarget upon photoactivation of the reactive group with light of theappropriate wavelength.

In a most preferred embodiment, these ligands are developed byphotoselection of nucleic acids (photoSELEX) (see U.S. Pat. No.5,763,177) and are capable of binding to a target. Upon photolysis theligands become covalently attached to the target. The addition of acovalent photocrosslink gives a secondary specificity, which is notnormally seen in diagnostics. In addition to binding, the formation of acovalent crosslink can only occur between the protein and the aptamer ifa chemically reactive electron donating amino acid is in proximity tothe photoaffinity label. This specificity is achieved by selection ofthe aptamer based on the ability to photocrosslink to the specifictarget protein if and only if a crosslinkable amino acid is in anorientation that is amenable to crosslink formation. Therefore, theaptamer will not crosslink to a protein that it was not specificallyselected to crosslink to even though it may be associated in anon-specific manner. The very covalent nature of the crosslink also addsto specificity in that it allows the crosslinked complex to be washedunder stringent conditions that would normally disrupt an affinity baseddetection. Stringent washing can therefore be used to decrease the noisein detection and thereby increase the ratio of signal to noise.

An array of photoreactive aptamers will be attached to biochip or othersurface, in a spatially defined manner, and then contacted with the testmixture. The chip can be directly irradiated, or gently washed beforeirradiation to remove the un-associated proteins. In effect, theirradiation will covalently attach only the correct protein to thecorrect photoactivitable aptamer presented at a defined area of a matrixlaid out on the surface of the chip. The protein, covalently bound tothe aptamer can be detected by sandwich assay, or fluorescent orradioactive protein dye as described in the above section entitled“Generic Detection Techniques”. The addition of covalent attachmentprovides that the detection of the protein can be achieved via chemicalmodification of reactive groups that are unique to the protein and notto the aptamer or chip. In addition, covalent attachment allows for amyriad of protein detection methods that are not limited by dissociationof the complex, such as organic solvents, temperature, denaturant orother methods that generally dissociate a non-covalent association.

Alternatively, the covalently associated complexes on the biochip willbe contacted with oligonucleotides complementary to all, or part of, thesequence of the nucleic acid ligand. Nucleic acid ligands that arecovalently bound to target will not be able to hybridize to thecomplementary oligonucleotide. The complementary oligonucleotides willbe labeled by any of the methods known in the art, as described above,to facilitate their detection.

V. Detection of Target Molecule Binding Through Methods That Do NotInvolve Fluorescence

Although preferred embodiments utilize fluorescent and phosphorescentdetection techniques for determining target molecule binding, there areother methods known in the art that have utility in this application.

A. Chemical Field Effect Transistors

Chemical field effect transistor (CHEM-FET) technology exploits thelocal change in chemical potential that is created upon the binding oftarget molecule to its ligand. In this technology, an insulative silica“gate” is placed between two n-type semiconductors, forming a biochip.Current will flow from one semiconductor to the other when a conductingchannel is formed in the gate and a potential difference is applied.Such channels will be opened when an ionic species binds to the silicagate (Schenk et al. U.S. Pat. No. 4,238,757). In another method (Lowe etal., U.S. Pat. No. 4,562,157), ligands are bound to discrete regions ofone of the semiconductors via photoactivation of derivatizing groups.The biochip is then contacted with a mixture containing targetmolecules. Binding of a target molecule to a ligand leads to a net lossor gain of ions at that location of the biochip. The ions locally alterthe conductance at this location, which in turn leads to a change in thedrain current in this area of the biochip. If the biochip is configuredin such a way that current drains will be measured in discrete locationson the biochip (multigated CHEM-FET), then spatial and quantitativeassessment of target binding will be achieved. Advances in the artshould permit the scaling up of this technology to independently andaccurately measure thousands of spatially discrete changes in draincurrent.

Another bioelectric change that can be measured using CHEM-FET is thephotoinduced electron transfer which occurs in double-stranded DNA(Murphy et al. (1993) Science 262:1025-1029). As mentioned above, thedegree of double-strandedness of each nucleic acid ligand may changewhen a target molecule is bound. Changes in the extent ofdouble-helicity will lead to localized changes in drain currents in aCHEM-FET biochip that is being illuminated. If the CHEM-FET biochip isread before and after contact with the target mixture, then detectingthese differences will reveal the sites and extent of target moleculebinding.

B. Detection Through Surface Plasmon Resonance

In a preferred embodiment, target molecule binding will be detectedthrough surface plasmon resonance (SPR). In this technique, nucleic acidligand is immobilized on a gold or silver film on the surface of aprism; the metal film is then incubated in the appropriate liquidmedium. Therefore, the metal film is at the prism-liquid interface.Light is directed through the prism towards the medium, and above acritical angle, total internal reflection of the light occurs. Abovethis critical angle, an evanescent wave extends into the medium by adistance that is approximately equal to the wavelength of the incidentlight. The evanescent wave excites free oscillating electrons, termedsurface plasmons, in the metal film, and causes them to resonate. Energyis absorbed from the evanescent wave by the electrons during thisprocess, thereby reducing the intensity of the internally reflectedlight. The angle at which total internal reflection, and henceresonance, occurs is exquisitively sensitive to changes in therefractive index of the medium immediately adjacent to the metal film.When a target molecule binds to a nucleic acid ligand on the surface ofthe film, the refractive index at this site changes, and the angleneeded to cause resonance changes also. Thus in order to detect targetmolecule binding, a detector system is arranged in which the angle ofincident light is varied, and the intensity of the reflected light ismeasured. Resonance occurs when the intensity of the reflected light isat a minimum. Measuring the change in angle of incident light needed tobring about resonance at specific sites on the film in the presence of atest mixture can then yield information about where a binding reactionhas occurred on the surface of the film. A device for measuring SPRcalled BIAcore7 is commercially available from Pharmacia Biosensors.

C. Detection Through the Use of Mass Spectrosocopy

In another embodiment, the formation of nucleic acid ligand-targetcomplex will be detected by mass spectroscopy. The surface of thebiochip will be irradiated in a spatially restricted and sequential wayusing a laser that is capable of ionizing the biological material on thebiochip. The mass of the ionization products will be detected by massspectroscopy, and comparison with the mass of ionization products of thesame unbound ligands will reveal where target is bound. This techniqueis known in the art as Matrix Absorption/Laser Desorption and Ionization(MALDI) Time of Flight Mass Spectroscopy. The nucleic acid ligands andthe targets in this embodiment can be covalently associated through theuse of photoactivatable crosslinking groups on the nucleic acid ligand,as described above in the section entitled “Detection of CovalentlyBound Target Molecules”.

D. Detection Through Atomic Force Microscopy (AFM) andScanning-Tunneling Microscopy (STM)

These related methods are well known in the art as techniques useful fordescribing the topology of surfaces at the nanometer level. Hence,advances in these techniques will make them suitable for detecting siteson a biochip where target molecule has been bound by a nucleic acidligand.

Atomic force microscopy (AFM) uses a non-metallic probe which is scannedover the surface of interest, in this case a biochip. The probe is movedclose to the surface so that the probe is subject to electron-repulsiveinteractions with the material bound to the surface. Repulsion leads tothe deflection of a cantilever upon which the probe is mounted, and thisdeflection is measured by a laser-photodiode detection system. Thesurface under examination is mounted on a stage, which stage is coupledto the deflection detection system by a computer. When the probe isdeflected, the stage is lowered, allowing the probe to trace out a“contour map” of electron density for the surface. Using this technique,a reference map for a nucleic acid biochip in a buffer will be prepared.This will be compared with a map obtained from a nucleic acid biochipthat has been incubated with a test mixture. Comparison of the two mapswill allow detection of sites on the biochip where target molecule hasbound.

Scanning tunneling microscopy (STM) uses a metallic probe which isscanned over a surface of interest. When the probe approaches thematerial bound to the surface, electrons can “tunnel” between the probeand the material, and the resulting current can be detected. The probeis scanned over the surface, and the vertical position of the probe isconstantly varied to permit tunneling. This, as in AFM, gives a map ofelectron density, which map will be used as described in the aboveparagraph to detect target molecule binding on a nucleic acid ligandbiochip.

VI. EXAMPLES

A. Example One

Nucleic acid ligand GB41 was isolated from a SELEX experiment againstthe U251 glioma cell line, as described in U.S. patent application No.08/434,425, filed May 3, 1995, now U.S. Pat. No. 5,789,157, entitled“Tissue SELEX.” Here, the nucleic acid ligand bears a 5′ biotin and isimmobilized to a streptavidin coated carboxylmethyl dextran biochipsurface (BIACORE 2000). The proteins are injected across flowcellscontaining GB41 or a version of GB41 in which the nucleotide sequence isscrambled. The scrambled sequence provides a test of binding specificityfor the nucleic acid ligand. Specific binding was detected tofull-length tenascin and to a bacterially expressed protein representingfibronectin type III repeats 3-5, which comprises 12% of the mass offull-length tenascin. These proteins did not bind to the scrambledsequence oligonucleotide. The slow dissociation of full-length tenascin,a hexamer, may result from multivalent interactions on the surface.Experiments established the association and dissociation rate constantsfor this protein-nucleic acid ligand interaction. The association phase(0-125 sec) was linear due to the large size (1.2 million dalton) oftenascin, which causes slow diffusion into the dextran matrix (masstransport-limited binding). The slow dissociation (125-300 sec) wasperhaps due to multivalent interactions that could form between thehexameric protein and the dextran-bound nucleic acid ligand.

B. Example Two

An NHS and an aldehyde reagent have been tested for their reactivitieswith proteins relative to nucleic acids. Fluorescein-NHS (MolecularProbes) was added to human serum albumin or alpha-1 HS glycoprotein (twoabundant plasma proteins) at 5000-fold molar excess. A 42-mer DNA wasalso added at 0-1000-fold molar excess relative to the protein. Thereaction was allowed to proceed for 30 min at room temperature at pH8.The protein, DNA and unreacted fluorophore were resolved by gelelectrophoresis, and the relative intensities of each product weredetermined by scanning and quantitation on a Molecular DynamicsFluorlmager. Fluorescein-NHS was 2.1×10⁴-fold more reactive with serumalbumin than with DNA on a mol:mol basis, and 4400-fold more reactive ona mass basis. Alpha-1 HS glycoprotein was 8000-fold more reactive thanDNA on a mol basis, and 3700-fold more reactive on a mass basis.

A similar experiment has been completed for the reaction of human serumalbumin with AttoTag CBQCA (Molecular Probes), an aldehyde couplingreagent which reacts with primary amines and cyanate to form afluorescent benzoisoindole product. In this case, the protein reactedreadily with albumin, but no reaction with DNA was detectable at a1000-fold excess of DNA over albumin. Albumin was calculated to be atleast 3×10⁴-fold more reactive to this reagent than is DNA.

What is claimed is:
 1. A biochip comprising an array of a plurality ofbiochip bound nucleic acid ligands, wherein a plurality of said nucleicacid ligands are specifically associated with a target molecule throughnon-Watson-Crick interactions.
 2. The biochip of claim 1, wherein eachof said nucleic acid ligands comprises a spectroscopically detectablelabel.
 3. The biochip of claim 2, wherein the spectroscopicallydetectable label is selected from the group consisting of a fluorescentlabel, a luminescent label, an energy transfer label, and aphosphorescent label.
 4. The biochip of claim 1, wherein each of saidnucleic acid ligands comprises a photoreactive group.
 5. The biochip ofclaim 1, wherein said target molecule is a protein.
 6. The biochip ofclaim 1, wherein the association between the nucleic acid ligand and thetarget molecule is covalent.
 7. The biochip of claim 1, furthercomprising a crosslinking agent, said crosslinking agent beingcovalently attached to the biochip, said each of said nucleic acidligands being attached to the crosslinking agent.
 8. The biochip ofclaim 1, wherein each of said biochip-bound nucleic acid ligands arebound to said biochip via a covalent bond, said covalent bond beingformed by the reaction of a first reactive group on said each of saidnucleic acid ligands, and a second reactive group on a surface of thebiochip.
 9. The biochip of claim 8, wherein said first reactive group isa thiol group, and said second reactive group is a thiol group.
 10. Thebiochip of claim 8, wherein said first reactive group is a 3′-hydroxylgroup, and said second reactive group is a nitroveratryl group.
 11. Thebiochip of claim 1, wherein each of said biochip-bound nucleic acidligands are bound to said biochip via a non-covalent association, saidnon-covalent association being formed by the interaction of a firstgroup on said each of said nucleic acid ligands, and a second group on asurface of the biochip.
 12. The biochip of claim 11, wherein said firstgroup is biotin, and said second group is streptavidin.
 13. The biochipof claim 1, wherein the target molecule comprises a detectable label.14. The biochip of claim 1, wherein the target molecule is associatedwith an antibody with a detectable label.
 15. The biochip of claim 1,wherein the target molecule is detectable by an antibody sandwich assay.16. The biochip of claim 1, wherein the target molecule is associatedwith a primary antibody, said primary antibody being associated with amolecule selected from the group consisting of a secondary antibody witha detectable label, a secondary antibody conjugated to a reporterenzyme, which enzyme catalyzes the production of a detectable compound,and protein A with a detectable label.
 17. The biochip of claim 1,wherein the target molecule is associated with a second nucleic acidligand selected from the group consisting of a nucleic acid ligand witha detectable label, a nucleic acid ligand with a fluorescent label, anda nucleic ligand conjugated to biotin.
 18. The biochip of claim 1,wherein the target molecule is detectable by a competition assay.